Subunit Redundancy Within the Nurd Complex Ensures Fidelity of ES Cell Lineage

Home , CHD3, MTA1, MTA2, MTA3

bioRxiv preprint doi: https://doi.org/10.1101/362988; this version posted July 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Subunit redundancy within the NuRD complex ensures fidelity of ES cell lineage

commitment

Thomas Burgold1,4, Michael Barber1, Susan Kloet2, Julie Cramard1, Sarah Gharbi1, Robin

Floyd1, Masaki Kinoshita1, Meryem Ralser1, Michiel Vermeulen2, Nicola Reynolds1, Sabine

Dietmann1 and Brian Hendrich1, 3

1. Wellcome– MRC Stem Cell Institute, University of Cambridge, Cambridge CB2 1QR

United Kingdom

2. Department of Molecular Biology, Faculty of Science, Radboud Institute for

Molecular Life Sciences, Radboud University, 6525 GA Nijmegen, The Netherlands

3. Department of Biochemistry, University of Cambridge, Cambridge CB2 1QR United

Kingdom

4. Present address: Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton,

Cambridge, CB10 1SA, United Kingdom

Corresponding Author:

Brian Hendrich, [email protected], +44 (0)1223 760205, @BDH_Lab

Key Words: NuRD, Chromatin, ES Cell, Lineage Commitment, Transcription

Abstract

Multiprotein chromatin remodelling complexes show remarkable conservation of

function amongst metazoans, even though components present in invertebrates are often

present as multiple paralogous proteins in vertebrate complexes. In some cases these

paralogues specify distinct biochemical and/or functional activities in vertebrate cells. Here

we set out to define the biochemical and functional diversity encoded by one such group of

proteins within the mammalian Nucleosome Remodelling and Deacetylation (NuRD)

complex: Mta1, Mta2 and Mta3. We find that, in contrast to what has been described in

somatic cells, MTA proteins are not mutually exclusive within ES cell NuRD and, despite

subtle differences in chromatin binding and biochemical interactions, serve largely

redundant functions. Nevertheless, ES cells lacking all three MTA proteins represent a

complete NuRD null and are viable, allowing us to identify a previously undetected function

for NuRD in maintaining differentiation trajectory during early stages of lineage

commitment.

2 bioRxiv preprint doi: https://doi.org/10.1101/362988; this version posted July 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Introduction

Mammalian cells contain a number of proteins capable of using ATP hydrolysis to shift

nucleosomes relative to the DNA sequence, thereby facilitating chromatin remodelling. In

mammals, these ATP-dependent chromatin remodelling proteins usually exist within

multiprotein complexes and play essential roles in the control of gene expression, DNA

replication and repair (Hargreaves and Crabtree 2011; Narlikar et al. 2013; Hota and

Bruneau 2016).

NuRD (Nucleosome remodelling and deacetylation) is one such multiprotein complex

which is unique in that it contains both chromatin remodelling and protein deacetylase

activity. NuRD is highly conserved amongst metazoans and has been shown to play

important roles in cell fate decisions in a wide array of systems (Denslow and Wade 2007;

Signolet and Hendrich 2015). For example, in embryonic stem (ES) cells NuRD controls

nucleosome positioning at regulatory sequences to finely tune gene expression (Reynolds et

al. 2012; Bornelöv et al. 2018) and in somatic lineages NuRD activity has been shown to

prevent inappropriate expression of lineage-specific genes to ensure fidelity of somatic

lineage decisions (Denner and Rauchman 2013; Knock et al. 2015; Gomez-Del Arco et al.

2016; Loughran et al. 2017). It was recently demonstrated that this is achieved in both ES

cells and B-cell progenitors by restricting access of transcription factors to regulatory

sequences (Liang et al. 2017; Loughran et al. 2017; Bornelöv et al. 2018). Additionally,

aberrations in expression levels of NuRD component proteins are increasingly being linked

to cancer progression (Lai and Wade 2011; Mohd-Sarip et al. 2017).

NuRD is comprised of two enzymatically and biochemically distinct subcomplexes: a

chromatin remodelling and a deacetylase subcomplex. The chromatin remodelling

subcomplex contains a nucleosome remodelling ATPase protein (Chd3/4/5) along with one

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of the zinc finger proteins Gatad2a/b and the Doc1/Cdk2ap1 protein, while the deacetylase

subcomplex contains class I histone deacetylase proteins Hdac1/2, the histone chaperones

Rbbp4/7, the Metastasis Tumour Antigen family of proteins, Mta1, Mta2 and Mta3 and, in

pluripotent cells, the zinc finger proteins Sall1/4 (Lauberth and Rauchman 2006; Allen et al.

2013; Kloet et al. 2015; Bode et al. 2016; Low et al. 2016; Miller et al. 2016; Spruijt et al.

2016; Zhang et al. 2016). These two subcomplexes are bridged by Mbd2/3, creating intact

NuRD. While HDAC and RBBP proteins are also associated with other chromatin modifying

complexes, the CHD, MBD, GATAD2 and MTA proteins are obligate NuRD components.

Functional and genetic data indicate that the CHD4-containing remodelling subunit may be

capable of functioning independently of intact NuRD (O'Shaughnessy and Hendrich 2013;

O'Shaughnessy-Kirwan et al. 2015), though it is not clear whether the deacetylase

subcomplex has any function outside of intact NuRD.

Changes in subunit composition in large multiprotein, chromatin modifying complexes

such as PRC1 and BAF has been shown to correlate with distinct changes in function to sites

of action in the chromatin in a cell-type specific manner (Ho and Crabtree 2010; Morey et al.

2012). The NuRD complex might therefore be expected to show similar diversity in both

composition and function and in fact, diversification of NuRD function has been described

through differential incorporation of different isoforms of NuRD component proteins

(Bowen et al. 2004). For example, Mbd2 and Mbd3 are mutually exclusive within NuRD (Le

Guezennec et al. 2006). While Mbd2 is not required for mammalian development, Mbd3 is

essential for early postimplantation mouse development (Hendrich et al. 2001).

Mbd2/NuRD is a methyl-CpG binding co-repressor complex which is dispensable for early

development but Mbd3/NuRD, a transcriptional modulator found at sites of active

transcription, has been shown to play important roles in regulation of cell fate decisions in

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multiple developmental systems (Feng and Zhang 2001; Reynolds et al. 2012; Gunther et al.

2013; Reynolds et al. 2013; Shimbo et al. 2013; Menafra et al. 2014). In brain development

NuRD complexes containing either CHD3, CHD4 or CHD5 play distinct roles during cortical

development (Nitarska et al. 2016).

Further functional and biochemical diversification occurs through alternate use of the

three MTA proteins within NuRD. MTA proteins function as a scaffold around which the

deacetylase subcomplex is formed, comprising a 2:2:4 stoichiometry of MTAs:HDACs:RBBPs

(Millard et al. 2013; Smits et al. 2013; Millard et al. 2016; Zhang et al. 2016). The three MTA

proteins are highly conserved, differing from each other predominantly at their C-termini.

The MTA1 protein was originally identified because of its elevated expression in metastatic

cell lines (Toh et al. 1994), and subsequently all three MTA proteins have been shown to be

up-regulated in a range of different cancer types (Covington and Fuqua 2014; Sen et al.

2014; Ma et al. 2016). The MTA1 and 3 proteins were shown to form distinct NuRD

complexes in breast cancer cells and in B-cells and were recruited by different transcription

factors to regulate gene expression (Mazumdar et al. 2001; Fujita et al. 2003; Fujita et al.

2004; Si et al. 2015). These studies did not detect biochemical interactions between MTA3

and the other MTA proteins, leading to the conclusion that MTA proteins are mutually

exclusive within NuRD. In contrast, Mta1 was shown to interact with Mta2 in MEL cells,

possibly indicating that mutual exclusivity may be cell type-specific (Hong et al. 2005). While

all three Mta genes are expressed in ES cells, detailed biochemical analysis of interactions of

MTA proteins with one another or with the various NuRD components in ES cells has not

previously been described.

Functional evidence does not support a strict lack of redundancy amongst MTA

proteins during mammalian development. While zygotic deletion of Chd4 or Mbd3 results in

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pre- or peri-implantation developmental failure respectively (Kaji et al. 2007;

O'Shaughnessy-Kirwan et al. 2015), mice deficient in any one of the three MTA proteins

show minimal phenotypes. Mice lacking either Mta1 or Mta3 are viable and fertile

(Manavathi et al. 2007)(Mouse Genome Informatics), while mice lacking Mta2 show

incompletely penetrant embryonic lethality and immune system defects (Lu et al. 2008). In

the current study we took a systematic approach to dissecting MTA protein biochemical and

functional diversity. We find that, in contrast to what has been described in somatic cells,

MTA proteins are not mutually exclusive within NuRD in ES cells and serve largely redundant

functions. Furthermore, ES cells lacking all three MTA proteins are viable and represent a

complete NuRD null, allowing us to identify a previously undetected function for NuRD in

early stages of lineage commitment.

Results

Mta proteins are not mutually exclusive within the NuRD complex in ES cells

The absence of a detected interaction between the MTA2 and MTA3 proteins in

human cells (Fujita et al. 2003; Si et al. 2015), and the observation that different MTA

proteins can show different protein-protein interactions in B-cells (Fujita et al. 2004) has led

to the conclusion that the MTA proteins are mutually exclusive within NuRD, and could

hence confer functional diversity to the NuRD complex (Lai and Wade 2011). To investigate

the biochemical specificity of the MTA proteins in an unbiased manner, we used gene

targeting of endogenous loci to produce three different mouse ES cell lines in which an

epitope tag was fused to the C-terminus of each MTA protein (Fig. 1A). Although MTA genes

show different expression patterns in preimplantation mouse development, all three are

expressed in peri-implantation and early postimplantation epiblast, the tissue most similar

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Burgold et al. Fig 1 A B Mta1- Mta3- Mta2- FLAG FLAG GFP Mta1 BAH ELM2 SANT GATA-ZnF TEV-Avi-3xFLAG 1 715

BAH ELM2 SANT GATA-ZnF GFP Mta2 1 IgG FLAG IP: IgG FLAG IP: IgG GFP IP: 668 Input Input Input 100 Mta3 BAH ELM2 SANT GATA-ZnF TEV-Avi-3xFLAG Mta1 1 591 Mta3 50

RBBPs 100 Mta2 Mbd GATAs 70 Sall 3/2 4 MTAs Chd4 50 Hdac2 HDACs Doc 1 Rbbp4 40 35 Mbd3 C

D Mta1 Mta2 Mta3 4 5 5 WT 4 4 -/- 3 Mbd3 3 3 2 2 2 rel to MTA rel to MTA rel to MTA 1 1 1

0 0 0 Chd4 Chd4 Chd4 Mbd2/3 Mbd2/3 Mbd2/3 Cdkap1 Cdkap1 Cdkap1 Rbbp4/7 Hdac1/2 Rbbp4/7 Hdac1/2 Rbbp4/7 Hdac1/2 Mta1/2/3 Mta1/2/3 Mta1/2/3 Gatad2a/b Gatad2a/b Gatad2a/b Sall1/2/3/4 Sall1/2/3/4 Sall1/2/3/4

Mta1Mta1 WT KO Mta Mta Mta1Mta1 WTMta1 KO SallMta1 SallWT KO Mbd Mbd Mta2Mta2 WT KO Mta Mta Mta2Mta2 WT KO SallMta2 Sall KO Mbd Mta3Mta3 WT KO Mta Mta Mta3Mta3 WTMta3 KO SallMta3 SallWT KO Mbd Mbd Mta1Mta1 WT KO Rbbp Rbbp Mta1Mta1 WT KO Chd4 Chd4 Mta2Mta2 WT KO Rbbp Rbbp Mta2 WT Mbd Mta2Mta2 WT KO Chd4 Chd4 Mta3Mta3 WT KO Rbbp Rbbp Mta3Mta3 WT KO Chd4 Chd4 Mta1Mta1 WT KO HDAC1/2 HDAC1/2 Mta1Mta1 WT KO Cdk2ap1 Cdk2ap1 Mta2 KO HDAC1/2 Mta2Mta2 WT KO Cdk2ap1 Cdk2ap1 Mta3Mta3 WT KO HDAC1/2 HDAC1/2 Mta3Mta3 WT KO Cdk2ap1 Cdk2ap1 Mta1Mta1 WT KO Gatad2a/b Gatad2a/b Mta2 WT HDAC1/2 Mta2Mta2 WT KO Gatad2a/b Gatad2a/b Mta3Mta3 WT KO Gatad2a/b Gatad2a/b Figure 1. Biochemical characterisation of epitope-tagged MTA proteins A. Schematic of MTA proteins with different protein domains indicated as coloured boxes, and the C-terminal epitope tags indicated.

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B. Tagged MTA proteins in heterozygously targeted cell lines were immunoprecipitated and subsequent western blots probed with antibodies indicated at right. Solid black triangles indicate the locations of untagged proteins, grey triangles show the position of epitope-tagged proteins, and open arrowhead show the locations of IgG bands in the final two lanes. Molecular weight in KDa are shown at left. (Full western blot images are available in Mendeley Data.) C. Proteins co-purifying with Mta1, Mta2 or Mta3 in IP-mass spectrometry experiments in wild type cells (top) or Mbd3-null cells (bottom). Proteins showing significant enrichment with the bait protein are located outside the dotted lines. For all panels the protein being immunoprecipitated is indicated in red, the other MTA proteins in purple, and other NuRD components in blue. Each IP/Mass spec experiment was carried out in biological triplicate. D. Relative enrichment of indicated proteins in MTA pulldowns from wild type (WT) or Mbd3-null (Mbd3KO) ES cells, normalised to 2x MTA proteins. NuRD components comprising the remodelling subunit are labelled in blue, those comprising the deacetylase subunit in red.

to the naïve ES cell state (Fig. S1). We therefore considered ES cells to be a good system in

which to investigate the function of MTA proteins.

Each tagged protein was expressed at levels comparable to those of wild type proteins

and was found to interact with other NuRD component proteins by immunoprecipitation

(Fig. 1B). Each MTA protein was also able to immunoprecipitate both of the other MTA

proteins in addition to unmodified forms of itself (Fig. 1B). Each NuRD complex contains two

copies of an MTA protein (Millard et al. 2013; Smits et al. 2013; Zhang et al. 2016), so the

identification of an interaction between MTA proteins means that individual NuRD

complexes in ES cells could contain either homodimers or heterodimers consisting of any

combination of the three MTA proteins.

To investigate the potential biochemical diversification conferred by different MTA

proteins in pluripotent cells we used our tagged cell lines to identify proteins interacting

with each of the MTA proteins by label-free quantitative mass spectrometry. Each protein

robustly co-purified with all known NuRD component proteins, including each of the MTA

proteins, confirming that MTA proteins are not mutually exclusive within NuRD in ES cells

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(Fig. 1C). In addition to NuRD components, each of the MTA proteins co-purified with Wdr5

as well as a number of zinc finger proteins, most of which had previously been identified as

NuRD-interacting proteins (Bode et al. 2016; Spruijt et al. 2016; Ee et al. 2017; Matsuura et

al. 2017).

As NuRD is assembled from a deacetylase subcomplex and a remodeller subcomplex

joined through the Mbd3 protein (Fig. 1A), loss of Mbd3 is expected to result in dissociation

of these two subcomplexes. Endogenous tagging for each Mta protein was performed in an

ES cell line harbouring a floxed Mbd3 allele, and IP/Mass spectrometry was repeated in ES

cells after Mbd3 deletion in order to enrich for interactions specific for the deacetylase

subcomplex. The majority of interactions with non-NuRD components was lost after Mbd3

deletion, indicating that most of these proteins do not directly associate with either the

MTA proteins or with the deacetylase subcomplex (Fig. 1C, Table S1). Exceptions to this

were Zfp296, which was identified as interacting with all three MTA proteins in an Mbd3-

independent manner, and Pwwp2a, which co-purified with Mta1 in both wild type and

Mbd3-null cells. Notably, Zfp219 and Zfp512b both showed Mbd3-independent interactions

specifically with Mta2 (Fig. 1C, Table S1).

By quantitating the abundance of peptides sequenced in each experiment we found

that the interactomes for both Mta1 and Mta2 showed a depletion of peptides associated

with the remodeller subcomplex (i.e. Chd4, Gatad2a/b and Cdk2ap1) in Mbd3-null cells,

whereas proteins associated with the histone deacetylase subcomplex (Mta proteins,

Hdac1/2, Sall proteins and Rbbp4/7) remained present at similar levels (Fig 1D). In contrast

the Mta3 interactome showed an increased interaction with Mbd2 and no relative loss of

either sub-complex in the absence of Mbd3. Thus both Mta1 and Mta2 can form part of a

stable deacetylase subcomplex in the absence of intact NuRD, but Mta3 is preferentially

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found in intact NuRD complexes. All three MTA proteins associated with Mbd2 in the

absence of Mbd3, indicating that they can all can contribute to Mbd2/NuRD. Together these

data show that the MTA proteins are found exclusively within the NuRD complex in ES cells.

MTA proteins show subtle differences in genome-wide binding patterns

To test whether MTA proteins confer differential chromatin binding to NuRD

complexes we subjected our ES cell lines expressing epitope-tagged MTA proteins to

chromatin immunoprecipitation followed by high throughput sequencing (ChIP-Seq). The

binding profiles of the three proteins were largely, but not completely overlapping (Fig. 2A).

Mta3 peaks were almost entirely associated with Mta1 and/or Mta2-binding, while 31% of

Mta1 peaks and 22% of Mta2 peaks were not associated with any other MTA protein (Fig.

2A). The vast majority of MTA peaks overlapped with Chd4 peaks, indicating that they

represent NuRD-bound regions (Figs. 2B, C). Mta3 peaks were almost exclusively associated

with Chd4 binding, consistent with Mta3 being preferentially in intact NuRD (Fig. S2A). In

contrast, 15% of Mta1 peaks and 10% of Mta2 peaks did not overlap with the Chd4 dataset.

Sites found associated with all three MTA proteins were highly enriched for Chd4 and

Mbd3 binding, consistent with these being core NuRD binding regions (Fig. 2C, top panels).

These sites were also enriched for marks of active promoters (H3K4Me3, H3K27Ac) and

active enhancers (H3K4Me1, H3K27Ac, P300; Fig. 2C, D), both of which are hallmarks of

NuRD-associated regions (Miller et al. 2016; Bornelöv et al. 2018). The same was true for

sites bound by any two of the three MTA proteins (Fig. S2B). The majority of sites occupied

by one MTA protein, but not the other two, were also occupied by Chd4 but to a lesser

extent than is seen at core NuRD sites (Fig. 2C, D). Whereas Mta2-only and Mta3-only sites

showed enrichment for both enhancer and promoter marks, Mta1-only sites showed no

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Burgold et al. Fig 2

A Mta1 B n=58798 Mta1 n=58798 18504 12683 Mta3 7059 n=22908 1238 88723 36953 2103 1427 18575 20481

3128 1668 10344 Mta2 Chd4 n=52528 11804 n=148703 Mta2 n=52528

C D

H3K4me1 Chd4 H3K4me3 H3K27ac Mbd3 Ep300

Colorkey Colorkey Colorkey Colorkey Colorkey Colorkey Colorkey Colorkey Colorkey

−3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 Mta1 Mta2 Mta3 Chd4 Mbd3 Ep300 H3K4me1 H3K27ac H3K4me3

Mta1 Mta2 Mta3 Chd4 Mbd3 p300 H3K4me1 H3K27ac H3K4me3 Mta1+2+3 Mta1/2/3 (n=18575)

Colorkey Colorkey Colorkey Colorkey Colorkey Colorkey Colorkey Colorkey Colorkey

−3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000

Mta1 Mta2 Mta3 Chd4 Mbd3 p300 H3K4me1 H3K27ac H3K4me3 Mta1 Only Mta1 Only (n=18504) Mta2 Only

Colorkey Colorkey Colorkey Colorkey Colorkey Colorkey Colorkey Colorkey Colorkey

−3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3

−3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 Mta1 Mta2 Mta3 Chd4 Mbd3 p300 H3K4me1 H3K27ac H3K4me3 Mta2 Only (n=11804)

Colorkey Colorkey Colorkey Colorkey Colorkey Colorkey Colorkey Colorkey Colorkey

Mta1 Mta2 Mta3 Chd4 Mbd3 p300 H3K4me1 H3K27ac H3K4me3 Mta3 Only

−3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 -3 0 3 -3 0 3 -3 0 3 -3 0 3 -3 0 3 -3 0 3 -3 0 3 -3 0 3 -3 0 3 Kb

-3 -2 0 2 3

log2(ChIP/Input) (n=1427) Mta3 Only

Figure. 2. Mta proteins show similar chromatin binding patterns A. Comparison of peaks identified by ChIP-seq for each MTA protein in wild type ES cells. Total peak numbers are indicated below each protein name. Each ChIP-seq dataset was made from biological triplicates. B. Comparison of Mta1 and Mta2 peaks with Chd4 peaks. C. ChIP-seq enrichment for indicated proteins or histone modifications is plotted across different subsets of Mta-bound sites. Mta1/2/3 refers to peaks identified in all three

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ChIP-seq datasets, whereas “Mta1 Only”, “Mta2 Only” or “Mta3 Only” refer to peaks only called for that protein. D. Average enrichment of density plots in C are plotted for different subsets of Mta ChIP-seq peaks.

enrichment for H3K4Me3, indicating that Mta1 is not found at promoters either without

one of the other Mta proteins or within the context of complete Mbd3/NuRD (Fig. 2C, D,

S2C). In contrast, sites bound by Mta2 and Mta3, but not Mta1 showed particular

enrichment for H3K4Me3 (Fig. S2B). None of the MTA-bound sequences showed any

enrichment for methylated DNA (Fig. S2D). In all cases MTA-only sites lacking Chd4, which

could represent sites of binding by the histone deacetylase subcomplex only, showed

characteristics of inactive enhancers, in that they were moderately enriched for H3K4Me1

and P300, but not for H3K4Me3, H3K27Ac or H3K36Me3 (Fig S2C). Genes associated with

binding by only one or two MTA proteins, with or without Chd4 are associated with a similar

distribution of GO terms as core NuRD sites (Fig S2E) which is not consistent with the idea

that the MTA proteins are directing NuRD activity to specific gene subsets.

Mta1/Mta2/Mta3 triple knockout is a total NuRD null

To identify specific functions for the different MTA proteins, we obtained gene trap

alleles for each of the three MTA genes from the European Conditional Mouse Mutagenesis

Programme (Skarnes et al. 2011) as ES cell lines (Mta1 and Mta2) or as embryos (Mta3) (Fig.

S3A-C). ES cell lines were used for morula aggregation to create chimaeric mice which were

subsequently outcrossed to wild type (C57Bl/6) mice to establish a mouse line. Conditional

deletion alleles were generated for each line, which was subsequently bred with females

expressing Sox2-Cre (Hayashi et al. 2002) resulting in deletion of floxed exons and the

absence of any detectable protein production from each allele (Fig. S3D; see Methods for

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Figure 3. Mta1/Mta2/Mta3 triple null ES cells represent a complete NuRD KO

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A. Western blots of wild type (Control), double or triple-null ES cell nuclear extract was probed with antibodies indicated at right. LaminB1 acts as a loading control. Approximate sizes are indicated in KDa. B. Phase-contrast images of wild type or Mta123∆ ES cells in self-renewing conditions. Scale bars represent 100µm. C. Western blots of anti-Chd4 immunoprecipitation of nuclear extract from indicated cell lines (top) probed with antibodies indicated at right. Approximate sizes are indicated in KDa. D. Western blot of a time course of Mta3 deletion in Mta1∆Mta2∆Mta3Flox/Flox: Cre-ER (Mta12∆3F) or Control (Mta12∆3F without Cre-ER) ES cells probed for Mta3, Mbd3 or Histone H3 as a control. The time course is indicated at the top as Days + tamoxifen. E. Western blot showing rescue of Mta123∆ ES cells by ectopic expression of Mta1, Mta2 or Mta3 from a transgene (TG). Total RNA Polymerase II acts as a loading control (RNAPII). For all western blots molecular weight is indicated at left in KDa. F. Model of NuRD complex structure. Upon loss of Mbd3 most of the NuRD complex falls apart into the Chd4-containing remodeller subcomplex and the MTA-containing deacetylase subcomplex, but some intact Mbd2-NuRD still remains. Upon loss of all three MTA proteins no intact NuRD remains, both Mbd3 and Gatad2a/b become unstable and neither of the intact subcomplexes remain.

details). As has been reported previously, Mta1-/- and Mta3-/- mice were viable, and Mta2-/-

mice showed incompletely penetrant embryonic lethality (Manavathi et al. 2007; Lu et al.

2008).

Mta1, Mta2 or Mta3-null ES cell lines derived from mice were morphologically

indistinguishable from wild type ES cells, as were ES cell lines deficient for combinations of

pairs of MTA genes (Fig. S3E). Mta1-/-Mta2-/-Mta3Flox/Flox ES cells (Mta12∆) were

subsequently created and expanded in culture (see Methods for details; Fig. 3A). After

transfection with a Cre expression construct to induce deletion of both Mta3 alleles we

recovered ES cells lacking all three MTA proteins. These Mta1-/-Mta2-/-Mta3-/- ES cells

(subsequently referred to as Mta123∆) appeared morphologically normal in standard, 2i +

LIF culture (Fig. 3B). MTA proteins are therefore dispensable for ES cell viability.

Structurally, MTA proteins bridge an interaction between the deacetylase subcomplex

with Mbd3 and the remodelling subcomplex (Fig. 1A) so we predicted that loss of all three

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MTA proteins would prevent NuRD formation. Consistent with this prediction we could

detect no interactions between Chd4 and components of the deacetylase subcomplex

(Hdac2, Rbbp4) in Mta123∆ ES cell nuclear extract by immunoprecipitation of endogenous

proteins (Fig. 3C). Surprisingly, despite being transcribed at normal levels, both Gatad2b and

Mbd3 proteins were barely detectable in Mta123∆ cells (Fig. 3C), indicating that the MTAs

are important for the stability of both of these proteins. To investigate this further we

monitored loss of Mta3 in Mta1∆/∆Mta2∆/∆Mta3Flox/Flox ES cells after deletion using a

tamoxifen-inducible Cre and found that loss of Mbd3 protein stability was coincident with

loss of Mta3 protein (Fig. 3D). Furthermore, introduction of either Mta1, Mta2 or Mta3 into

Mta123∆ ES cells at levels comparable to wild type expression resulted in restoration of

Mbd3 protein levels, demonstrating that contact with at least one of the MTA proteins is

sufficient for Mbd3 protein stability (Fig. 3E). In contrast to Mbd3-null ES cells which display

a significant depletion, but not a complete loss of NuRD due to partial compensation by

Mbd2 (Kaji et al. 2006), Mta123∆ ES cells were completely devoid of any detectable intact

NuRD (Fig. 3C, F). The Mta123∆ ES cells are thus a total NuRD null ES cell line, which allowed

us to examine, for the first time, the consequences of a complete loss of the NuRD complex

in a viable mammalian cell system.

The NuRD complex safeguards cellular identity during differentiation

Twice as many genes showed an increase rather than a decrease in expression levels

in Mta123∆ ES cells compared to control ES cells by RNA-seq (Fig. 4A). This is consistent with

NuRD acting predominantly, but not exclusively, as a transcriptional repressor. The ratio of

increased to decreased gene expression in Mta123∆ cells was very similar for genes bound

by all three Mta proteins, Mta1+2, Mta1 only or Mta2 only (Fig. 4B), indicating that NuRD’s

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Figure 4. Mta proteins act redundantly to control gene expression A. Comparison of gene expression in Mta123∆ ES cells and wild type (WT) ES cells. Each circle represents a gene: red indicates spike-in controls, blue indicates genes that are

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not differentially expressed to a significant degree, and green indicates differentially expressed genes (2404 increased, 1293 decreased) defined with an adjusted p-value < 0.05 and a log2 fold-change > 1. N=3 for each genotype. B. Fold change in gene expression is plotted for different subsets of genes. All genes are plotted in black, while subsets of genes located nearest to ChIP-seq peaks for the indicated proteins +Chd4 and which show significant changes in expression compared to wild type cells are plotted in dashed coloured lines as indicated. The number of genes in each of the Mta categories are: all genes (32271), all differentially expressed (n=3701), Mta1+2+3 (n=1738), Mta1+2 (n=1460), Mta1 (n=1020) and Mta2 (n=924). C. Principal component analysis of RNAseq data from ES cell lines of indicated genotypes in either self-renewing conditions (2i) or after 48 hours in the absence of two inhibitors and LIF (Diff48). Each point represents a biological replicate. D. Expression of indicated genes segregated by functional category for wild type (WT, green circles), Mbd3∆ (blue triangles), or Mta123∆ (magenta squares). Data are taken from RNA-seq experiments and each point represents the merging of two biological replicates each for 1 (Mbd3∆) or two (WT, Mta123∆) independently derived cell lines. E. Phase contrast pictures of ES cells of indicated genotypes after 5 days in differentiation conditions (N2B27). Scale bars represent 100 µm. F. Expression of indicated genes was measured by RT-qPCR in wild type (Control, red), Mbd3∆ (purple) or Mta123∆ (blue) ES cells over a time course of differentiation. N ≥ 3 biological replicates, error bars indicate SEM. G. Genes associated with indicated GO terms plotted by fold change in expression in Mta123∆ ES cells (x-axis) or Mbd3∆ ES cells (y-axis) induced to differentiate for 48 hours. Genes are coloured if they are differentially expressed (log2 fold-change > 1 and padj value < 0.05) in either comparison as indicated. The dotted lines show the fold-change cut-off of 2. GO-terms were identified using David v.6.8 (Huang da et al. 2009) using a Benjamini score with a cutoff of 0.05.

impact on transcription is not detectably altered by the inclusion or absence of individual

MTA subunits. Sites bound by MTA proteins in the absence of Chd4, and presumably

representing sites bound by the deacetylase subcomplex, were not associated with specific

gene misexpression events in the Mta123∆ ES cells (Fig. S4A, B).

Comparing global gene expression profiles using principal component analysis (PCA)

showed that Mta123∆ and Mbd3∆ ES cells in self-renewal conditions were more similar to

each other than either was to wild type cells (Fig. 4C). Mta123∆ ES cells were most distinct

from wild type cells, consistent with them representing a more complete NuRD knockout

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(Fig. 4C, S4C). Consistent with this, genes driving the NuRD-specific separation (Principal

Component 2; PC2) were generally misexpressed to a greater degree in Mta123∆ ES cells

than in Mbd3∆ ES cells (Fig 4D, S4C). As has been shown for Mbd3∆ ES cells, both NuRD

mutant lines moderately misexpressed pluripotency-associated genes in ES cells (Reynolds

et al. 2012)(Fig. 4D). Yet in 2iL conditions pluripotency-associated genes were mis-expressed

to a much lesser extent than genes associated with differentiation (Fig. 4D). This

demonstrates that, in addition to maintaining pluripotency gene expression levels, NuRD

also functions in ES cells to prevent inappropriate activation of lineage-specific gene

expression.

Despite showing inappropriate expression of a large number of genes normally

associated with differentiated cells, Mta123∆ ES cells could be maintained as

morphologically undifferentiated ES cells in the very restrictive 2iL conditions (Fig. 3B). We

therefore next asked what impact complete loss NuRD activity had upon the differentiation

capacity of Mta123∆ ES cells. Upon removal of the two inhibitors and LIF from the culture

media wild type cells began to adopt the flatter morphology of neurectoderm (Fig. 4E)(Ying

et al. 2003). This was accompanied by downregulation of pluripotency-associated genes and

the activation of a neural gene expression programme (Fig. 4F, S4C). Mbd3∆ ES cells are able

to respond to the absence of self-renewal factors but have a very low probability of

adopting a differentiated fate when induced to differentiate in N2B27 conditions (Kaji et al.

2006; Reynolds et al. 2012). Consistent with these findings, after 5 days in differentiation

conditions Mbd3∆ ES cells showed some signs of having responded to differentiation

conditions, but still retained pockets of morphologically undifferentiated cells (Fig. 4E). In

contrast the completely NuRD-null Mta123∆ ES cells appeared to have all exited the self-

renewal programme and adopted a flat, monolayer morphology (Fig. 4E). The ability of

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Mta123∆ ES cells to undergo morphologically normal neuroectodermal differentiation was

rescued upon re-expression of either Mta1, Mta2 or Mta3 (Fig. S5A).

Since the differentiation process may be considered as a combination of exit from self-

renewal (downregulation of pluripotency-associated genes) and acquisition of lineage

specific gene expression programs, we focussed on changes to these two classes of genes by

RT-qPCR over a differentiation time course. Pluripotency-associated genes such as Esrrb,

Klf4, Nanog and Pou5f1 were downregulated during differentiation of both wild type and

Mta123∆ ES cells (Fig. 4F). While this downregulation was not absolutely dependent on the

presence of functional NuRD, the magnitude and kinetics of the response varied in a gene-

dependent manner. Genes associated with acquisition of neural fate were activated in the

presence of wild type NuRD, a response which was reduced in the NuRD null line (Fig. 4F).

Globally, Mta123∆ ES cells misexpressed considerably more genes associated with

development and differentiation than did Mbd3∆ ES cells after 48 hours of differentiation

(Fig. S4E). Exogenous expression of either Mta1, Mta2 or Mta3 in Mta123∆ ES cells was able

to rescue the ability of cells to activate neural gene expression to different extents during

differentiation (Fig. S5B), consistent with their ability to rescue the morphological

phenotypes. Mta123∆ ES cells induced to differentiate towards a mesoderm fate similarly

failed to appropriately activate differentiation markers, and exogenous expression of

individual MTA proteins was again able to rescue the defect to varying degrees (Fig. S5C).

Together these data show that while NuRD activity is not required for ES cells to exit the

naïve state, it is required for activation of lineage-appropriate gene expression as well as to

prevent expression of genes associated with other cell types.

To better understand how NuRD-null cells respond when induced to differentiate we

compared their gene expression profiles to a transcription landscape made from single cells

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Burgold et al. Fig 5 A

DAPI Cdx2 Sox2 B DAPI Cdx2 Sox2 K-Orange K-Orange K-Orange K-Orange Control Δ Mta123

C **** **** **** **** 15 15 8

6 10 10 * 4

5 5 Caspase+ 2 Cells/Blastocyst

Orange cells/embryo 0 0

0 Orange & Sox2/embryo Mta123∆ Control Mta123∆ Control Mta123∆ Control Mta123∆ Control Mta123∆ + Mta2TG N = 18 N = 52 N = 18 N = 52 N = 16 N = 39 N = 18 N = 52 N = 22 Orange+ Orange+ Sox2+ Sox2- Caspase D DAPI Caspase K-Orange K-Orange Δ Mta123

Figure 5. NuRD activity maintains an appropriate ES cell differentiation trajectory A. Comparison of expression data for wild type, Mbd3∆ or Mta123∆ ES cells in self- renewing (2i) conditions or after 48 hours in differentiation conditions (Diff48) with

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mouse embryonic single cell RNA-seq data from (Mohammed et al. 2017). Larger circles represent biological replicates, smaller circles represent individual cells. PC4 vs PC1 is plotted in Fig. S5D. B. Composite images of representative chimaeric embryos made with control (Mta1Flox/∆Mta2+/+Mta3Flox/Flox) or Mta123∆ ES cells. ES-derived cells express the Kusabira Orange fluorescent marker. Sox2 indicates epiblast cells and Cdx2 is expressed in trophectoderm cells. Arrows indicate examples of K-Orange expressing cells in the mutant embryos. Scale bars = 20µm. C. (Left) Number of K-Orange expressing cells observed in chimaeric embryos obtained using control ES cells, Mta123∆ ES cells, or Mta123∆ ES cells in which Mta2 was reintroduced on a constitutively expressed transgene (Mta123∆+Mta2TG). P-values calculated using a two-tailed t-test. (Middle) Mean number of K-Orange cells per embryo separated by Sox2 expression. (Right) Number of K-Orange and Caspase-3 positive cells per embryo. P-values calculated using a one-tailed t-test: *P < 0.05, ****P < 0.0001, “ns” = not significant. D. Composite images of representative chimaeric embryos as in Panel B stained with an anti-activated Caspase 3 antibody. Arrows indicate an example of an orange, apoptotic cell. Scale bars = 20µm.

taken from early mouse embryos (Fig. 5A; S5D) (Mohammed et al. 2017). In the self-

renewing state, Mbd3D and Mta123D cells clustered near control (parental) lines near

embryonic day 3.5 (E3.5) and E4.5 inner cell mass cells, as is expected for naïve mouse ES

cells (Boroviak et al. 2014). After 48 hours of differentiation control ES lines clustered with

E5.5 epiblast cells, demonstrating that the ES cell differentiation process occurred

analogously to development in vivo. While Mbd3D ES cells appear to have exited the self-

renewing state and to have taken the same differentiation trajectory as wild type cells (i.e.

leftwards along PC1; Fig 5A, S5D), they did not travel as far along this trajectory as wild type

cells, instead occupying a space between the E4.5 and E5.5 epiblast states. Mta123D cells

travelled even less far along PC1 than Mbd3∆ ES cells, and rather than maintain the

appropriate differentiation trajectory they also travelled along PC2, occupying a space

between E4.5 epiblast and E4.5 primitive endoderm. This further demonstrates that not

only is NuRD important for cells to be able to adopt the appropriate gene expression

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programme for a given differentiation event, but it is also important for cells to maintain an

appropriate differentiation trajectory.

If Mta123∆ ES cells were undergoing a specific trans-differentiation event towards

trophectoderm during ES cell differentiation then this could become more pronounced if

exposed to normal differentiation conditions in a chimaeric embryo. If, in contrast, they are

simply unable to differentiate properly, they would not be expected to contribute to early

embryos. To distinguish between these possibilities, we assessed the ability of Mta123∆ ES

cells to differentiate in chimaeric embryos. Equal numbers of control or Mta123∆ ES cells

expressing a fluorescent marker were aggregated with wild type morulae and allowed to

develop for 48 hours. While wild type cells contributed to the ICM of host embryos with

100% efficiency, Mta123∆ ES cells showed significantly reduced contribution and increased

levels of apoptosis (Fig. 5B-D). Those Mta123∆ cells that did survive in blastocysts were

predominantly, but not always found in the inner cell mass and expressed Sox2 but not

Cdx2, indicating that they had not undergone inappropriate differentiation towards a

trophectoderm fate. This is most consistent with Mta123∆ cells being unable to properly

adjust to an ICM environment and enter a normal differentiation path. The ability to

contribute to the ICMs of chimaeric embryos was rescued by constitutive expression of

Mta2 in Mta123∆ cells (Fig. 5C). We therefore propose that NuRD functions not only to

establish the correct lineage identity of cells during the differentiation process, but also

prevents inappropriate gene expression to maintain an appropriate differentiation

trajectory (Fig. 6).

22 bioRxiv preprint doi: https://doi.org/10.1101/362988; this version posted July 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Burgold et al. Fig 6

NuRD identity exit from pluripotency NuRD

Mbd3D

exit from NuRD pluripotency identity

Mta123D NuRD

exit from identity pluripotency

Figure 6. Model of NuRD function during differentiation of pluripotent cells. NuRD facilitates lineage commitment of ES cells after exit from pluripotency (red arrows), allowing cells to form differentiated cell types (Top). In the absence of Mbd3, residual NuRD activity ensures cells retain the appropriate differentiation trajectory, but the cells are unable to reach a differentiated cell fate (dotted red arrows; Middle). In the absence of all three MTA proteins there is no residual NuRD activity and ES cells are unable to either achieve appropriate lineage commitment, or to maintain the proper differentiation trajectory (dotted arrows, bottom panel).

Discussion

Here we provide a biochemical and genetic dissection of the core NuRD component

MTA proteins in mouse ES cells. In contrast to what has been found in somatic cell types,

23 bioRxiv preprint doi: https://doi.org/10.1101/362988; this version posted July 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

MTA proteins are not mutually exclusive in ES cell NuRD complexes and all combinations of

Mta homo- and hetero-dimers can exist within NuRD. Different MTA proteins exhibit subtle

differences in chromatin localisation or biochemical interaction partners, but we find no

evidence for protein-specific functions in self-renewing or differentiating mouse ES cells. ES

cells completely devoid of MTA proteins are viable but show inappropriate expression of

differentiation-associated genes, are unable to maintain an appropriate differentiation

trajectory and do not contribute to embryogenesis in chimaeric embryos.

Protein subunit diversity is often found in chromatin remodelling complexes which

specifies functional diversity (Hargreaves and Crabtree 2011; Morey et al. 2012; Hota and

Bruneau 2016). This is also the case for the NuRD complex, where alternate usage of

Mbd2/3, Chd3/4/5, and Mta1/3 has been found to result in alternate functions for NuRD

complexes (Feng and Zhang 2001; Fujita et al. 2003; Nitarska et al. 2016). Yet our findings

that Mta1 and Mta3-null mice are viable and fertile, and that we detect no major

differences in the abilities of different MTA proteins to rescue the Mta123∆ ES cell

phenotypes indicates that MTA proteins exhibit considerable functional redundancy.

Different MTA proteins are capable of interacting with each other in ES cells, so how the

mutual exclusivity reported in other cell types might be achieved is not clear. One possibility

is that the variable inclusion in NuRD of zinc finger proteins, such as the Sall proteins in ES

cells, could influence the MTA makeup of NuRD complexes. This class of variable NuRD

interactors, which include Sall1/2/3/4, Zfp423 (Ebfaz), Zfpm1/2 (Fog1/2), and Bcl11b,

interact with RBBP and/or MTA proteins via a short N-terminal motif (Hong et al. 2005;

Lauberth and Rauchman 2006; Lejon et al. 2011). Of this class of proteins Sall1 and Sall4 are

the most highly expressed in ES cells, and Sall4 can associate with all three MTA proteins

(Fig. 1C) (Miller et al. 2016). In contrast the Zfpm1 (Fog1) protein was shown to

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preferentially associate with Mta1 and Mta2, but not Mta3, in a somatic cell line (Hong et al.

2005). Hence it is possible that different proteins using this N-terminal motif to interact with

NuRD in different cell types could act to skew the proportion of different MTA proteins

included in the NuRD complex.

Mta1 and Mta2 both contain two distinct RBBP interaction domains, while Mta3 lacks

the C-terminal most RBBP interaction domain (Millard et al. 2016). The Mta1 and Mta2

proteins show minimal loss of stability in the absence of Mbd3, but Mta3 requires an

interaction with Mbd3 to be completely stable (Bornelöv et al. 2018). It is possible that the

additional interaction with an Rbbp protein confers stability to Mta1 and Mta2 in the

absence of Mbd3. This would be consistent with our interpretation that Mta3 preferentially

exists within an intact NuRD complex (Figs 1, 2). Rbbp4/7 confer histone H3 binding to the

NuRD complex, so Mta3-containing NuRD may bind chromatin less tightly than Mta1-

and/or Mta2-containing NuRD. Consistent with this possibility, we identified >2x more

Mta1- and Mta2-assoicated ChIP-seq peaks than Mta3-peaks (Fig. 2A).

Pluripotent cells lacking Mbd3 have been used extensively to show that NuRD plays

important roles in control of gene expression during early stages of exit from pluripotency in

vitro and in vivo (Kaji et al. 2006; Kaji et al. 2007; Latos et al. 2012; Reynolds et al. 2012).

Mbd3-null ES cells contain Mbd2/NuRD and thus represent a NuRD hypomorph, rather than

a NuRD-null. ES cells lacking all three MTA proteins show no detectable NuRD formation

(Fig. 3C) and therefore we propose represent a true NuRD null. Mta123∆ ES cells are similar

to Mbd3∆ ES cells in that both misexpress a range of genes in 2iL conditions and both fail to

properly undergo neuroectodermal differentiation. Yet the Mta123∆ ES cells misexpress a

larger number of genes than do Mbd3∆ cells in both self-renewing and differentiation

conditions, and they show a more pronounced differentiation defect. We propose that the

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increased transcription of a wide array of lineage inappropriate genes in Mta123∆ ES cells

renders them incapable of not only achieving the correct differentiation state, but also of

maintaining the proper differentiation trajectory (Fig. 6). In this model the primary function

of NuRD in ES cells is to silence inappropriate gene expression, which ensures fidelity of

differentiation. A further, more specialised function contained within the core function and

dependent upon Mbd3 (but not Mbd2), is to respond properly to context-appropriate

differentiation signals to achieve specific differentiation states.

Materials and Methods

Mouse embryonic stem cells

Mouse embryonic stem cells (ESCs) were grown on gelatin-coated plates in 2i/LIF

conditions as described (Ying et al. 2008). The following “Knockout First” alleles were

obtained from EUCOMM as heterozygous ES cell lines (Illustrated in Fig. S1A, B):

Mta1tm1a(EUCOMM)Wtsi

https://www.mousephenotype.org/data/alleles/MGI:2150037/tm1a(EUCOMM)Wtsi

Mta2tm1a(EUCOMM)Wtsi

https://www.mousephenotype.org/data/alleles/MGI:1346340/tm1a(EUCOMM)Wtsi

ES cells were used to derive mouse lines by blastocyst injection using standard

methods. ESC derivation was performed by isolating ICMs and outgrowing in 2i/LIF ESC

media as described (Nichols et al. 2009).

The epitope tagged ESC lines Mta1-Avi-3×FLAG and Mta2-GFP were generated by

traditional gene targeting, while the Mta3-Avi-3×FLAG line was generated using a

CRISPR/Cas9 genome editing approach. All Mta epitope tagged lines were made in an

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Mbd3Flox/- background (Kaji et al. 2006). Transient expression of Dre recombinase was then

used to remove the selectable marker (Anastassiadis et al. 2009).

ES cells were induced to differentiate towards a neuroectoderm fate by removal of

two inhibitors and LIF and culturing in N2B27 media as described (Ying et al. 2008).

Mesendoderm differentiation was performed as follows: ES cells were plated at 104

cells/cm2 in N2B27 on fibronectin-treated 6-well plates and cultured for 48 hours. Medium

was then replaced with 10 ng/ml activin A and 3 µM CHIR99021 in N2B27 and cultured

further.

Mice

All animal experiments were approved by the Animal Welfare and Ethical Review Body

of the University of Cambridge and carried out under appropriate UK Home Office licenses.

The Mta3 “Knockout First” allele was obtained from EUCOMM as a heterozygous mouse line

(Illustrated in Fig. S1C):

Mta3tm3a(KOMP)Wtsi

https://www.mousephenotype.org/data/alleles/MGI:2151172/tm3a%2528KOMP%25

29Wtsi?

Heterozygous knockout first mouse lines were crossed to a Flp-deletor strain kindly

provided by Andrew Smith (University of Edinburgh) (Wallace et al. 2007) to generate

conditional alleles. Mice harbouring conditional alleles were crossed to a Sox2-Cre

transgenic line (Hayashi et al. 2002) to create null alleles.

Chimaeric embryos were made by morula aggregation with 8-10 ES cells per embryo

as described (Hogan et al. 1994) and cultured for 24-48 hours prior to fixation and

immunostaining. ES cells used stably expressed a PiggyBac Kusabira Orange transgene.

27 bioRxiv preprint doi: https://doi.org/10.1101/362988; this version posted July 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Immunoprecipitation

Antibodies were incubated with Protein G-Sepharose beads (Sigma) for 1h at room

temperature. Nuclear extract (200 µg) was diluted in IP-Buffer (50 mM Tris-HCl pH 7.5, 150

mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol) with protease inhibitors and

incubated with antibody-bead conjugates at 4 °C overnight. Beads were washed in Low Salt

Wash-Buffer (IP-Buffer containing 150 mM NaCl), followed by High Salt Wash-Buffer (IP-

Buffer containing 300 mM NaCl). Antibodies are listed in Table S1. Full images of original

western blots are available on Mendeley Data: doi:10.17632/sxg8d5sgv6.1.

Label-free pulldowns and label-free quantitation (LFQ) LC-MS/MS analysis

Label-free pulldowns were performed in triplicate as previously described (Kloet et al.

2016; Miller et al. 2016). The mass spectrometry proteomics data have been deposited to

the ProteomeXchange Consortium via the PRIDE partner repository with the dataset

identifier PXD009855.

Chromatin immunoprecipitation (ChIP), sequencing, and analysis

Chromatin immunoprecipitations were carried out as previously outlined (Reynolds et

al. 2012). For sequencing of ChIP DNA, samples from six (Mta1-FLAG and Mta2-GFP) and

four (Mta3-FLAG) individual ChIP experiments were used. Antibodies used are listed in Table

S1. ChIP-seq libraries were prepared using the NEXTflex Rapid DNA-seq kit (Illumina) and

sequenced at the CRUK Cambridge Institute Genomics Core facility (Cambridge, UK) on the

Illumina platform. Reads were aligned using bowtie version 0.12.8 and the arguments -m 1.

Peaks were called using macs2 version 2.1.0 with the default Q-value of < 0.05. The results

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were merged to get 7 different sets of peaks defined by the combination of bound Mta

proteins.

DNA Methylation data was obtained from (Shirane et al. 2016). Output methylation

files were filtered for base pairs with a coverage of over 3. The methylation percentage was

averaged using a sliding window of 250 bp to give a smoothed track for the

heatmaps/profile.

High throughput sequence datasets used in this manuscript are listed in Supplemental

Table 2.

Gene expression analyses

Total RNA was purified using RNeasy Mini Kit (Qiagen) including on-column DNase

treatment. First-strand cDNA was synthesized using SuperScript IV reverse transcriptase

(Invitrogen) and random hexamers. Quantitative PCR (qPCR) reactions were performed

using TaqMan reagents (Applied Biosystems) on a QuantStudio Flex Real-Time PCR System

(Applied Biosystems) or a StepOne Real-Time PCR System (Applied Biosystems). Gene

expression was determined relative to housekeeping genes using the ΔCt method. TaqMan

assays are listed below.

Gene TaqMan assay Ascl1 Mm03058063_m1 Atp5a1 Mm00431960_m1 Cdh2 Mm01162497_m1 Cdx2 Mm01212280_m1 Esrrb Mm00442411_m1 Eomes Mm01351985_m1 Elf5 Mm00468732_m1 Foxa2 Mm01976556_s1 Gata3 Mm00484683_m1 Hes6 Mm00517097_g1 Klf4 Mm00516104_m1

29 bioRxiv preprint doi: https://doi.org/10.1101/362988; this version posted July 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Nanog Mm02019550_s1 Nestin Mm00450205_m1 Pax6 Mm00443081_m1 Pou5f1 Mm03053917_g1 Ppia Mm02342430_g1 Sox1 Mm00486299_s1 Sox17 Mm00488363_m1 T Mm00436877_m1 Tbp Mm00446971_m1 Zfp42 Mm03053975_g1

RNA-seq

Libraries for sequencing were prepared using the NEXTflex Rapid Directional RNA-seq

kit (Illumina) or SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara

Bio) and sequenced on the Illumina platform as for ChIP-seq libraries. Reads were aligned

using tophat version v2.1.0 (Kim et al. 2013) to genome build GRCm38/mm10. Gene

expression was quantified using featurecount version 1.5.0 (Liao et al. 2014) with

annotation from Ensembl release 86 (Yates et al. 2016). Normalization and differential

expression were performed using Deseq2 version 1.14.1 (Love et al. 2014), using R version

3.3.3. Deseq size factors were calculated using RNA spike-ins.

Acknowledgments

We thank Bill Mansfield, Peter Humphreys, Maike Paramor, Vicki Murray, and Sally

Lees for technical assistance and advice, and A. Smith, E. Laue and members of the BDH lab

for discussions and comments. Funding to the BH and MV labs was provided through EU FP7

Integrated Project “4DCellFate” (277899). The BH lab further benefitted from a Wellcome

Trust Senior Fellowship (098021/Z/11/Z) and from core funding to the Cambridge Stem Cell

Institute from the Wellcome Trust and Medical Research Council (097922/Z/11/Z and

203151/Z/16/Z).

30 bioRxiv preprint doi: https://doi.org/10.1101/362988; this version posted July 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

TB and BH devised the study; TB, SK, SG, RF, NR and BH generated the data; MB, MR

and SD analysed high throughput sequencing data, SK and MV generated and analysed

proteomics data, MK provided methodology and NR and BH wrote the manuscript with

input from other authors.

References

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35 bioRxiv preprint doi: https://doi.org/10.1101/362988; this version posted July 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Supplemental Data

Burgold et al. Fig S1

Mta1 1000 Mta2 Mta3

100

10 FPKM values

0.1 E2.5 E3.5 E4.5 Epi E4.5 PE E5.5 Epi ES cells

Figure S1. Expression of MTA genes during early mouse development RNAseq data from (Boroviak et al. 2014) plotted at indicated days of mouse development for each of the MTA genes. All data points are shown, with horizontal bars indicating the mean.

36 bioRxiv preprint doi: https://doi.org/10.1101/362988; this version posted July 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Burgold et al. Fig S2 A B

Colorkey Colorkey Colorkey Colorkey Colorkey Colorkey Colorkey Colorkey Colorkey

−3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3

Mta1 Mta2 Mta3 Chd4 Mbd3 p300 H3K4me1 H3K27ac H3K4me3

Mta1/2

Colorkey Colorkey Colorkey Colorkey Colorkey Colorkey Colorkey Colorkey Colorkey

−3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3

Colorkey Colorkey Colorkey Colorkey Colorkey Colorkey Colorkey Colorkey Colorkey

−3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 −3 −2 0 2 3 Mta2/3 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 Mta1 Mta2 Mta3 Chd4 Mbd3 p300 H3K4me1 H3K27ac H3K4me3

Mta1/3 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 −3000 −1500 Center 1500 3000 -3 0 3 Kb

-3 -2 0 2 3 log2(ChIP/Input)

C Mta1 Only Mta2 Only Mta3 Only + Chd4 - Chd4 + Chd4 - Chd4 + Chd4 - Chd4 (n=11619) (n=6885) (n=8758) (n=3046) (n=998) (n=429) FC

H3K4me3 2 Log

H3K36me3 FC 2 Log

H3K4me1 FC

H3K27ac 2

EP300 Log

Chd4 FC 2

Mbd3 Log

D E cell death regulation of cell proliferation actin cytoskeleton organization cell motility locomotion

% % Methylation metabolic process regulation of metabolic process regulation of signal transduction cell differentiation system development developmental process

1 10 100 -Log(P)

Mta1/Chd4 (6749) Mta1 (4339)

% % Methylation Mta2/Chd4 (7277) Mta2 (2430) Mta1+2/Chd4 (14752) Mta1+2 (1651)

Figure S2. Chromatin features of MTA-bound peaks (related to Figure 2). A. Comparison of ChIP-seq peaks for Mta3 and Chd4. The number of Mta3-only, Chd4- only, or Mta3+Chd4 peaks are indicated.

37 bioRxiv preprint doi: https://doi.org/10.1101/362988; this version posted July 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

B. Features of peaks containing two of the three MTA proteins, as in Figure 3C. C. Average enrichment of indicated features is shown for peaks for each Mta protein which do or do not also contain Chd4. The number of peaks in each set is indicated as n. D. Average enrichment of DNA methylation across MTA peaks with or without Chd4, as in Panel C. E. Significance of GO-term enrichment for genes associated with peaks for different combinations of Mta proteins and Chd4.

38 bioRxiv preprint doi: https://doi.org/10.1101/362988; this version posted July 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

A Mta1tm1a(EUCOMM)Wtsi Burgold et al. Fig S3

FRT FRT loxP loxP lacZ neo 1 SA pA 2 3 LacZ Reporter/KO + FLP FRT loxP loxP

1 2 3 Conditional + Cre FRT loxP

1 3 KO B Mta2tm1a(EUCOMM)Wtsi

FRT loxP FRT loxP loxP lacZ neo 1 2 3 SA pA 4-13 14 LacZ Reporter/KO + FLP

FRT loxP loxP

1 2 3 4-13 14 Conditional + Cre loxP

1 2 3 14 KO

tm3a(KOMP)Wtsi C Mta3 FRT FRT loxP loxP lacZ neo 1 2 3 SA pA 4 5 LacZ Reporter/KO + FLP FRT loxP loxP

1 2 3 4 5 Conditional + Cre

FRT loxP

1 2 3 5 KO

D KO KO KO E Control Mta1KO Mta12∆ Mta1 Mta2 Mta3 Control

260 αRpb1 NTD 70 αMta1

Mta2KO Mta3KO Mta13∆ 260 αRpb1 NTD

70 αMta2 70 αMta3

Figure S3. MTA reporter and knockout alleles (related to Figure 3) A. Schematic of the Mta1 “Knockout First” reporter allele Mta1tm1a(EUCOMM)Wtsi (Top). Exons are depicted as boxes, with normal coding exons as filled boxes. Exons around the insertion site are numbered. Coding exons not able to be translated in the

39 bioRxiv preprint doi: https://doi.org/10.1101/362988; this version posted July 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

depicted allele are shaded in light blue. The targeting resulted in an FRT-flanked LacZ-Neo fusion protein being expressed from the endogenous Mta1 promoter and preventing transcription of most Mta1 exons. Recombination between FRT sites is achieved by expression of FLP recombinase (middle), removing the LacZ-Neo cassette and restoration of Mta1 coding potential. Subsequent recombination between LoxP sites by Cre recombinase (bottom) results in loss of Exon 2 and subsequent exons are out of frame. B. Schematic of the Mta2tm1a(EUCOMM)Wtsi allele as in panel A. In this allele the neo gene is expressed from a human ß-actin promoter. In this allele expression of Cre results in excision of exons 4-13. C. Schematic of the Mta3tm3a(KOMP)Wtsi allele as in panel A. D. Western blot of nuclear extracts made from each single mutant (i.e. after Cre- mediated recombination) probed with indicated antibodies. Anti-Rbp1 NTD (RNA polymerase II subunit) acts as a loading control. E. Phase-contrast images of cell lines of indicated genotype in self-renewing conditions. Scale bars indicate 100 µm.

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Burgold et al. Fig S4 A B 15000

10000 DE All Genes 5000 Number of Genes

Mta1 Mta2 Mta1+2 Mta1+2+3 Mta1 NuRDMta2 NuRD Mta12 NuRD Mta123 NuRD

D Differentially expressed in Mta123Δ 2iL vs WT 2iL

extracellular extracellular matrix organisation matrix organization sodium sodium ion ion transport transport regulation regulation of membrane potential of membrane potential potassiumpotassium ion transmembrane ion transport transport cell fate commitmentcell fate commitment cell adhesioncell adhesion cell differentiationcell differentiation ion ion transport transport multicellular multicellular organism organism development development transmembrane transmembrane transport transport

0 −2 −4 −6 −8 −10

log10 (log10(Benjamini)Benjamini)

Differentially expressed in Mbd3Δ 2iL vs WT 2iL

positive regulation positive regulation of cell of proliferation cell proliferation

positive regulationtranscription of transcription from from RNAPII promoter RNA polymerase II promoter

multicellular multicellular organism organism development development

0.0 −0.5 −1.0 −1.5 −2.0 −2.5 0.0 -0.5 -1.0 log10(Benjamini)-1.5 -2.0 -2.5 log10 (Benjamini)

Genes not significantly differentially expressed Significantly differentially expressed in: Mbd3∆ Mta123∆ Mbd3∆ + Mta123∆

Figure S4. Control of gene expression by the MTA proteins (Related to Figure 4). A. Same as Fig. 5B, but plotting genes associated with Mta peaks lacking Chd4 enrichment.

41 bioRxiv preprint doi: https://doi.org/10.1101/362988; this version posted July 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

B. Plot showing the number of genes associated with indicated classes of Mta ChIP- peaks which do (red) or do not (black) show a significant change in expression in Mta123∆ ES cells in 2iL. C. Comparison of gene expression changes in wild type, Mbd3-null (Mbd3∆) and Mta123 triple-null (Mta123∆) ES cells in self-renewing (2i) conditions. The top 100 genes contributing to PC2 from Figure 5C are shown. “Mta123 WT” indicates a control cell line derived at the same time as the Mta123∆ cells. D. GO term enrichment for genes differentially expressed in the indicated comparisons. Significant gene ontology terms are plotted by log10 of the Benjamini-adjusted p- value. The significant GO terms and p-values were calculated using David v.6.8 E. Genes associated with indicated GO terms plotted by fold change in expression in Mta123∆ ES cells (x-axis) or Mbd3∆ ES cells (y-axis) as compared to wild type cells. Each point is a gene that has been annotated with that GO-term. Genes are coloured if they are differentially expressed in either comparison or both, using a log2 fold- change > 1 and a padj value < 0.05. The dotted lines are at the fold-change cut-off of 2.

42 bioRxiv preprint doi: https://doi.org/10.1101/362988; this version posted July 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Burgold et al. Fig S5

A Mta123∆+ Mta123∆+ Mta123∆+ WT Mta123∆ Mta1 TG Mta2 TG Mta3 TG

5D N2B27

B Esrrb Pou5f1 Nanog Klf4 Control Mta123∆ + Mta1TG 10 10 10 10 Mta123∆ + Mta2TG Mta123∆ Mta123∆ + Mta3TG 1 1 1 1

0.1 0.1 0.1 0.1 Relative Expression Relative Expression Relative Expression 0.01 Relative Expression 0.01 0.01 0.01

0 4 days 0 4 days 0 4 days 0 4 days Nestin Pax6 Ascl1 Cdh2 100 100 100 100

10 10 10 10 1 1 1 1 0.1 0.1 Relative Expression Relative Expression Relative Expression Relative Expression 0.1 0.01 0.01 0.1 0 4 days 0 4 days 0 4 days 0 4 days Cdx2 Eomes Elf5 Gata3 100 10 10000 1000 1000 100 10 100 1 10 10 1 1 1 Relative Expression Relative Expression Relative Expression 0.1 0.1 Relative Expression 0.1 0.1

0 4 days 0 4 days 0 4 days 0 4 days C

Foxa2 1000 Foxa2 1000 Control Mta123∆ + Mta1TG 100 100 Mta123∆ + Mta2TG Mta123∆ 10 10 Mta123∆ + Mta3TG Relative Expression Relative Expression 1 1 0 1 2 3 4 days differentiation 0 4 days Sox17 100 Sox17 100

10 10 D

1 1 Relative Expression Relative Expression 0.1 0.1 0 1 2 3 4 days differentiation 0 4 days T 100 T 1000

100 10 10 1 1 Relative Expression Relative Expression 0.1 0.1 0 1 2 3 4 days differentiation 0 4 days Klf4 10 Klf4 10

1 1 0.1 Relative Expression Relative Expression 0.01 0.1 0 1 2 3 4 days differentiation 0 4 days

Zfp42 10 Zfp42 10

1 1 Relative Expression Relative Expression 0.1 0.1 0 1 2 3 4 days differentiation 0 4 days

Figure S5. Failure of differentiation in Mta123∆ ES cells (related to Figures 5 and 6). A. Phase contrast images of ES cells of indicated genotypes induced to differentiate for 5 days in N2B27. B. Comparison of gene expression in indicated ES cell lines in undifferentiated conditions or after 4 days differentiation in N2B27. qPCR was carried out in triplicate

43 bioRxiv preprint doi: https://doi.org/10.1101/362988; this version posted July 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

at each time point for a minimum of three biological replicates. Error bars indicate SEM. C. Comparison of gene expression in indicated ES cell lines induced to differentiate towards mesoderm. qPCR was carried out in triplicate at each time point for a minimum of three biological replicates. Error bars indicate SEM. D. Same as Figure 6A, plotting PC4 vs PC1.

Table S1. Antibodies used in this study

Antibody Company WB IP IF ChIP (Product Number) anti-MTA1 Cell Signaling 1:2000 - - (5647) anti-MTA2 Abcam 1:5000 3 µg - (ab50209) anti-MTA3 Proteintech 1:2000 - - (14682-1-AP) anti-MBD3 Abcam 1:5000 - - (ab157464) anti-CHD4 Abcam 1:5000 5 µg - (ab70469) anti-HDAC2 Santa Cruz 1:1000 - - (sc-7899) anti-RBBP4 Abcam 1:5000 - - (ab79416) anti-GATAD2B Bethyl Laboratories 1:1000 - - (A301-281A) anti-Histone H3 Abcam 1:5000 - - (ab1791) anti-RNA Pol II Santa Cruz 1:2000 - - subunit RPB1 (sc-899X) anti-FLAG M2 Sigma-Aldrich 1:2000 3 µg 10µg (F1804) anti-GFP Abcam 1:5000 15µg 25µg (ab290) anti-Sox2 e-biosciences 1:500 (14-9811-82) anti-CASPASE-3 Cell Signalling 1:500 cleaved (9664S) anti-Cdx2 Abcam 1:250 (ab157524)

Table S2. High-throughput sequencing datasets used in this study.